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Institut de Biologie StructuraleGrenoble / France

Contact person(s) related to this article / Eric Forest

Hydrogen/deuterium exchange – mass spectrometry

Presentation

CTC autosampler and Agilent UHPLC in the back of the Agilent mass spectrometer

Hydrogen/deuterium exchange mass spectrometry (MS) enables a rather fine identification (5-10 amino acids) of regions of (soluble or membrane) proteins implied in conformational changes or interactions with partners.

It enables in particular to map the stability of an antibody and to identify epitopes.

The technique involves incubation in a deuterated buffer of a protein in different forms or in presence and absence of its partner, to exchange some amide hydrogens with deuterium. After trapping the exchanged deuteriums and proteolysis of the protein, the mass of the different peptides is measured using MS.
-  Conformational changes: for a peptide from the protein under different forms, the mass differences indicate the differences of deuterium incorporation and thus local conformational changes;

-  Interface identification: a mass decrease for a peptide from the protein between the free and the complexed form indicates that the region of this peptide is implied in the interface with the partner.

The technique has been automated recently, from the sample processing to the data processing.

HDX of peptides using BmrA 3-D models

3-D models of BmrA in the open conformation (left side) or in the closed conformation (right side). One subunit was drawn in wheat colour and the other in light grey. Identified peptides are drawn with rainbow colours (in one monomer and in all ICDs) according to their percentage of deuterium exchange after 3600 s (scale of exchange shown on the right).
Mehmood, S., Domene, C., Forest, E. & Jault, J.-M. (2012). Dynamics of a bacterial multidrug ABC transporter in the inward and outward facing conformations. Proc. Natl. Acad. Sci. USA 109, 10832-10836.

Key words

Hydrogen/deuterium exchange; mass spectrometry; antibody; epitope; interaction; conformation.

Specific equipment

- CTC autosampler
- Peltier-cooled box for valves and columns
- Agilent HPLC pumps
- Agilent electrospray mass spectrometer
- Sierra Analytics HDExaminer software

Access

The public research groups may access to this platform while answering to a call for proposals (ANR, EU, etc) and/or with a specific person implied in the study.
The industrial laboratories may access through a contract, with respect for confidentiality (NDA signature).

Samples

A study requires some hundreds of μg of pure homogeneous protein at a minimum concentration of 5 μM and lasts from some weeks to some months.
The glycosylated proteins must have their glycosylations identified or be fully deglycosylated.
Membrane proteins can be studied if they are soluble in DDM. Other nonionic detergents (Triton X100, polyoxyethylene-based, PEG, PPG) can be also envisionned.

Results

Collaborative project: the results are used to be included into publications with coauthorship of implied people.

Company: the results are detailed in a confidential report.

Contact

For any information on the possibility, the necessary means and the setup of a study, contact Eric Forest.

Publications

For an overview of the projects studied at the IBS with this technique, here are:

- Some publications :

Mehmood, S., Domene, C., Forest, E. & Jault, J.-M. (2012). Dynamics of a bacterial multidrug ABC transporter in the inward and outward facing conformations. Proc. Natl. Acad. Sci. USA 109, 10832-10836.

Rey, M., Man, P., Clemencon, B., Trezeguet, V., Brandolin, G., Forest, E. & Pelosi, L. (2010). Conformational Dynamics of the Bovine Mitochondrial ADP/ATP Carrier Isoform 1 Revealed by Hydrogen/Deuterium Exchange Coupled to Mass Spectrometry. Journal of Biological Chemistry 285, 34981-34990.

Marcoux, J., Man, P., Castellan, M., Vives, C., Forest, E. & Fieschi, F. (2009). Conformational changes in p47(phox) upon activation highlighted by mass spectrometry coupled to hydrogen/deuterium exchange and limited proteolysis. Febs Letters 583, 835-840.

Man, P., Montagner, C., Vernier, G., Dublet, B., Chenal, A., Forest, E. & Forge, V. (2007). Defining the interacting regions between apomyoglobin and lipid membrane by hydrogen/deuterium exchange coupled to mass spectrometry. Journal of Molecular Biology 368, 464-472.

Brier, S., Lemaire, D., DeBonis, S., Forest, E. & Kozielski, F. (2004). Identification of the protein binding region of S-trityl-L-cysteine, a new potent inhibitor of the mitotic kinesin Eg5. Biochemistry 43, 13072-13082.

Cravello, L., Lascoux, D. & Forest, E. (2003). Use of different proteases working in acidic conditions to improve sequence coverage and resolution in hydrogen/deuterium exchange of large proteins. Rapid Communications in Mass Spectrometry 17, 2387-2393.

- The list of the IBS publications describing the developments and the applications of the technique.